PROJECT SUMMARY/ABSTRCT Multidrug-resistant organisms (MDRO) are major threats to human health, responsible for >2 million infections and 23,000 deaths per year in the US alone. Urinary tract infections (UTI) are the most common types of infections caused by MDROs. There are few options to prevent and treat MDRO UTIs, no interventions to reverse fecal MDRO colonization, and limited strategies to identify patients at risk for recurrence. This study will address critical barriers to scientific progress in the fields of recurrent MDRO UTI and fecal MDRO colonization, and improve scientific knowledge by delineating the host microbial communities and MDRO traits that are associated with MDRO colonization and recurrent UTI. To advance my patient-oriented research career and pursue additional expertise in genomic research, I will conduct a prospective cohort trial of patients with MDRO UTIs and characterize their stool and urine specimens utilizing microbiologic culture and genomic techniques. We will assess longitudinal changes in the fecal microbiome of patients with MDRO UTI utilizing metagenomic shotgun sequencing to identify microbial taxa and functions associated with persistent MDRO colonization and recurrent UTI, and link this information with detailed clinical data. The proposals delineated here will advance the understanding of the microbial ecology of the fecal microbiome and its role in MDRO infections, and will provide me the opportunity to gain knowledge and skills in study implementation, longitudinal data analysis, and genomic data analysis. I have assembled a team of mentors and collaborators to guide my early research career and advise on the evaluation of existing stored specimens created through a pilot study, and ongoing recruitment of patients for the prospective cohort trial. We will analyze the specimens to answer the following questions: 1) What is the trajectory of the fecal microbiome and resistome in patients with MDRO UTIs after antimicrobial treatment? 2) Are there taxonomic and functional compositions of the fecal microbiome associated with MDRO colonization and recurrent infection? 3) How does antimicrobial treatment impact the concentration of antibiotic resistance genes and bacterial virulence factors, and what is their relationship to recurrent MDRO UTI? Genomic approaches and classic microbiologic techniques will be used to assess the strain types, microbial taxa, function, and antibiotic resistance genes found in the fecal microbiome of patients with MDRO UTIs. The culmination of these studies will provide essential data to drive the rational design of methods to detect and prevent fecal MDRO colonization and MDRO UTIs. The findings in this study will be broadly applicable beyond UTIs, as we will determine the relationship between fecal MDRO colonization and recurrent infections.